Diamine acid fluorescent chelates

ABSTRACT

Species-linked diamine triacetic acids of the formula ##STR1## wherein T is an organic species containing at least one amine, hydroxyl, or thio functional group, L is the residue of at least one of those functional groups and R is a two or more atom long covalent bridge, are disclosed. Methods for their preparation, for the preparation of metal chelates from them and for the use of the chelates are also disclosed. In a preferred embodiment, the metal ions employed in the formation of the chelates are rare earth metal ions capable of forming fluorescent chelates which can in turn be employed in fluoroassay techniques.

This is a division of application Ser. No. 73,728, filed Sept. 10, 1979,now U.S. Pat. No. 4,352,751.

FIELD OF THE INVENTION

The subject of this invention is a diamine triacetic acid capable offorming metal chelates. More particularly, this invention relates to abifunctional ligand useful to bind metal ions to organic species such asorganic target molecules or antibodies, preferably to formspecies-linked fluorescent rare earth metal chelates useful influorescent assay techniques.

BACKGROUND OF THE INVENTION

Fluorescence techniques are finding increasing application in chemicaland biochemical and medical analyses. Fluorescence measurement methodsare intrinsically extremely sensitive. They can offer at least thesensitivity of radiochemical methods without the hazards associated withradiation.

U.S. Pat. Nos. 4,150,295 and 4,058,732, issued on Apr. 17, 1979 and Nov.15, 1977, respectively, and a chapter appearing at pages 67-80 ofImmunofluorescence and Related Staining Techniques, Knapp, et al eds.(1978, Elsevier/North Holland Biomedical Press) disclose the generalconcept of fluorescently quantitating nonfluorescent species usingfluorophores with long decay lifetimes in comparison with ambientfluorescence. In this method, an atomic scale fluorescent tag(fluorophore) is chemically covalently affixed to the individualmolecules of a species. This species may be an organic target moleculeitself or it may be a molecule essentially identical with a targetmolecule or it may be an antibody specific to a target molecule. Aftersuitable procedures, dependent on the form of the assay, the taggedspecies are excited and, using time-gated techniques taught in the threereferences, their fluorescence measured. Using the magnitude of theobserved fluorescence and a previously preparedfluorescence/concentration standard curve, the amount of target isdetermined.

Fluorophores and tagged species useful in such a determination ideallyhave a long fluorescent decay lifetime and retain their ability tofluoresce throughout the period of the analysis. For sensitive assays itis also important that the fluorophore and its linkage to other speciesbe stable even at very low concentrations such as in the range ofnanograms/cc and lower such as even to femtograms/cc. For manyimmunoassays it is desirable that the fluorophore be water soluble so asto be reacted with antibodies in an environment which preserves immunereactivity.

The metal chelates of the present invention include within their numbera family of species-bound ligands which possess all of these desiredproperties. In addition, they are simple and inexpensive to prepare.

STATEMENT OF THE INVENTION

Stated in its broadest sense, our invention is that an organic species(herein denominated as "T") having at least one of the functional groups##STR2## can be attached to one, and essentially only one, of the fourcarboxyl groups of diamine tetraacetic acids forming a stable structurepossessing the configuration as shown in General Formula I wherein R isa two or more atom long covalent bridge and L is the deprotonatedequivalent of said functional group on the species molecule, T. ##STR3##Since T may contain more than one L group, more than one diaminetriacetic acid may be so covalently linked within this formula.

These covalently linked products (Formula I.) are referred to as"species-linked diamine triacids". They form stable chelates withvarious metal ions. These chelated complexes (Formula II.) constituteanother aspect of this invention ##STR4## wherein "M" is a metal ioncapable of forming a coordination complex with diamine tri- ortetraacids. In a preferred embodiment, the metal ion, M, is an ion of arare earth metal capable of forming a fluorescent chelate complex.

In a third aspect of this invention, a particularly favorable complex isformed when an activator such as a salicylic acid is also present in aternary complex combination with the rare earth metal ion and thespecies-linked diamine triacid.

In a fourth aspect, this invention provides a process for determiningthe concentration or presence of an organic species, T, such as a targetmolecule or an antibody that contains at least one of the groups##STR5## The process involves contacting such a species molecule with asubstantial excess of a diamine tetraacetic acid dianhydride of GeneralFormula III, wherein R is a two or more atom long covalent bridge,##STR6## under conditions to effect reaction of at least one of saidgroups on the species molecule with one of the anhydride groups of thedianhydride to yield a species-bound diamine triacid anhydride;hydrolyzing the remaining anhydride group to yield the species-linkeddiamine triacid; admixing said species-linked diamine triacid insolution with a rare earth metal ion to form a species-linked rare earthchelate; then, optionally after suitable procedures depending on thetype of assay involved, flooding this species-linked rare earth chelatewith an activator such as a salicylic acid; measuring the intensity offluorescence of said fluorescent complex; and relating the intensity ofthe observed fluorescence to the intensity of fluorescence of a knownconcentration of such a fluorescent complex.

In a fifth aspect, this invention provides a process for detecting thepresence or amount of a target molecule by coupling one or moretetraacid dianhydride molecules of Formula III to ##STR7## sites on anantibody specific to a target molecule to form an antibody with one ormore diamine triacid molecules covalently linked thereto; forming a rareearth chelate of said antibody coupled triacid; exposing a quantity ofthe coupled antibody to tissue, liquid, or solid substrate suspected ofcontaining the target molecule to bind said target to the antibody,removing excess unbound antibody, flooding the liquid, tissue, or solidsubstrate with a fluorescent activator specific to the rare earthtriacid chelate and measuring or detecting the fluorescence as anindicator of the quantity or presence of said target molecules.

DETAILED DESCRIPTION OF THE INVENTION

This Description will be divided into the following sections:

The Species-Linked Diamine Triacid Compounds,

Metal Complexes,

Ternary Combinations with Flooders,

Preparative Methods,

Species Molecules, and

Analysis Techniques.

THE SPECIES-LINKED DIAMINE TRIACID COMPOUNDS

These compounds have the structure set forth in General Formula I. InFormula I, R is a two or more atom covalent bridge. The two or moreatoms refer to the atom in the bridge itself. They may be substitutedwith additional atoms or groups if desired. The function of R is tocovalently bond together the two amine diacetic acid groups in a spacingwhich permits the two amine diacetic acid groups to form a stablechelate with metal ions. Thus, any group that will serve this functionwithout interfering with the chelate-forming ability of the aminediacetic acid groups can be employed as R.

Preferably, R is an optionally substituted two to eight atom covalentbridge selected from carbon-oxygen ether bridges, carbon-nitrogenpolyalkyl secondary or tertiary amide bridges and carbon-carbon bridgesincluding alkylenes, cycloalkylenes, or arylenes, all eitherunsubstituted or containing substituents pendant from the bridge.Substituents include, for example, alkyls of from one to about tencarbon atoms, aryls of from six to ten carbon atoms, aralkyls andalkaryls of from seven to about fourteen carbon atoms. The bridges orthe aforementioned substituents may also be substituted with carboxyls,carbonyls, ethers, carbamates, secondary amides, sulfonates, sulfamates,and the like. Exemplary R groups include ethylene, n-propylene,isopropylene, the various butylenes including n-butylene, 1- and2-methylpropylene, 1-propylethylene, 1-cyclohexylethylene,1-phenylethylene, alkyl-substituted 1-phenylethylenes and propylenes,1-benzylethylene, 2-amidopropylene, cyclohexyl-1,2-ene, phenyl-1,3-ene,the diethylene-ether --CH₂ --CH₂ --O--CH₂ --CH₂ --, the triethylenediether --CH₂ --CH₂ --O--CH₂ --CH₂ --O--CH₂ --CH₂ --, and the like. Thislist is not intended to be inclusive but merely to represent typicalembodiments of group R.

For reasons of simplicity of preparation, unsubstituted alkylenes offrom two to five and preferably two to four carbons in length arepreferred R's with ethylene and propylene being more preferred andethylene being the most preferred R.

The species-linked diamine triacid compounds of this invention containone or more functionalities, L, which are a primary or secondary amidenitrogen, an ester oxygen or a thioester sulfur. Each unit of L servesas a covalent linkbetween one unit of the diamine ligand and the speciesmolecule, T. The choice of L depends upon the nature of the speciesmolecule as L may be derived from an amine (primary or secondary), ahydroxyl or a thiol present on the species molecule. In general, theamide nitrogens are the preferred L groups with the amide group (thusderived from a primary amine group on T) being the most preferred.

The species-linked diamine triacid compounds of the invention containtwo amine groups, one of which carries two acetic acid groups and theother of which carries one acetic acid group and one acetic acid/L-Tadduct. For purposes of simplicity, the three acetic acid groups aredepicted in their protonated form throughout this specification andclaims. It will be appreciated that in actuality these groups, beingweak acids, exist in equilibrium between the protonated form shown andthe corresponding deprotonated salt form, --COO⁻. The exact proportionsof the two forms depend upon the pH and composition of the environmentof use. It is intended that the protonated form shown shall representthe equilibrium of the two forms which actually exists.

METAL COMPLEXES

The species-linked diamine triacids of this invention are effectivechelating ligands. It appears that their complex-forming ability issimilar if not essentially the same as that of the correspondingnon-species-linked diamine tetraacetic acid ligands (such asethylenediamine tetraacetic acid - EDTA). It further appears that thefourth carbonyl group, although used in the attachment to the species,may be able to function as a point of coordination with metal ions andthus that the dissociation constants of complexes formed with thediamine triacids of this invention are very similar to those obtainedwith the diamine tetraacetic acids of the art.

The metal ions, M, which can be complexed include ions ofradionucleotides as well as nonradioactive metal ions. Although notextensively proven, it appears that essentially all of the art-knowncomplexes of metal ions with EDTA and its non-species-linked analogs canbe prepared using the species-linked ligands of this invention. Forexample, complexes can be formed with M equal to ions of the transitionmetals as well as the rare earth metals of both the actinide andlanthanide series. Exemplary metal ions represented by M include Al⁺⁺⁺,Am⁺⁺⁺, Cd⁺⁺, Ce⁺⁺⁺, Cf⁺⁺⁺, Cm⁺⁺⁺, Co⁺⁺, Co⁺⁺⁺, Cr⁺⁺, Cr⁺⁺⁺, Cu⁺⁺, Dy⁺⁺⁺,Er⁺⁺⁺, Eu⁺⁺⁺, Fe⁺⁺, Fe⁺⁺⁺, Ga⁺⁺⁺, Gd⁺⁺⁺, Hg⁺⁺, Ho⁺⁺⁺ , In⁺⁺⁺, La⁺⁺⁺,Lu⁺⁺⁺, Mn⁺⁺, Mn⁺⁺⁺, Nd⁺⁺⁺, Ni⁺⁺, Pb⁺⁺, Pd⁺⁺, Pm⁺⁺⁺, Pr⁺⁺⁺, Pu⁺⁺⁺,Pu⁺⁺⁺⁺, Sb⁺⁺⁺, Sc⁺⁺⁺, Sm⁺⁺⁺, Sm⁺⁺, Sn⁺⁺, Tb⁺⁺⁺, Th⁺⁺⁺⁺, Ti⁺⁺⁺, Tl⁺⁺⁺,Tm⁺⁺⁺, V⁺⁺⁺, V⁺⁺⁺⁺⁺, VO₂ ⁺, Y⁺⁺⁺, Yb⁺⁺⁺, Zn⁺⁺, and Zr⁺⁺⁺⁺.

Because of the scope of the possible metal ions to be incorporated andthe wide range and diversity of properties that such metal ions exhibit,the metal ion complexes in accordance with this invention findapplication and utility throughout the field of chemical analysis. Thepresence of the metal ions linked by the present invention to thespecies molecule can be detected by such techniques as radioassay, X-rayscattering or fluorescence, NMR or ESR shifts, or the like, dependingupon the metal ion incorporated. In a preferred embodiment, the metalions employed are those rare earth metal ions which form complexes withEDTA type chelating ligands. Preferred among these are terbium,dysprosium, europium, samarium, and neodimium with terbium and europiumbeing more preferred and terbium being the most preferred rare earth forforming metal ion M.

The complexes formed between the metal ion, M, and the species-linkeddiamine triacid is considered to be a 1:1 equimolar metal:chelatecomplex. It is represented structurally by the structure given asGeneral Formula II.

TERNARY COMBINATIONS WITH FLOODERS

The preferred application of the species-linked diamine triacids of thisinvention is to employ them as chelating ligands in fluorescent rareearth metal complexes. The utility of these complexes in fluorescenceanalytical techniques has been found to be substantially enhanced when athird component is present in the complex. This third component, apromoter, is generically referred to as a "flooder" because it isusually added in large excess. Flooders, which are also referred to aspromoters or sensitizers, increase the fluorescence excitationefficiency of the rare earth chelates and have been disclosed innon-species-linked rare earth ions and chelates. See Dagnall et al,ANALYST, 92, 358-363, (1967); Heller and Wasserman, J. CHEM. PHYS., 42,949-955, (1965); McCarthy and Winefordner, ANAL. CHEM., 38, 848, (1966);Taketatsu et al, TALANTA, 13, 1081-1087, (1966); Alberti et al, ANAL.CHEM., 38, 214-216, (1966) and Charles et al, J. INORG. NUCL. CHEM., 28,527-536, (1966). These references and their disclosure of usefulflooders are incorporated herein by reference. This is not intended as acomplete list but is suggestive of the large number of flooding agentsthat can be used with the species-linked diamine triacids of thisinvention.

Many of these flooders appear to have coordination properties and to actby occupying sites on the rare earth metal ion that are not alreadyoccupied by the species-linked diamine triacid ligand. In so doing, theybecome closely coupled with the metal ion allowing for efficient energytransfer from the flooder to the rare earth metal.

Examples of complex-forming flooders are 5-sulfosalicylic acid (5-SSA),4-aminosalicylic acid, salicylic acid, other substituted salicylicacids, dipicolinic acid, and the like. These materials are especiallyuseful with terbium complexes, which application demonstrates theiraction.

A complex of a species-linked diamine triacid and terbium exhibits anexcitation band near 2400 Å. This is a relatively weak band and, at thiswave length, is not at a convenient part of the spectrum. When 5-SSA isadded as a flooder a strong excitation band is observed in the nearultraviolet, near 3100 Å, a much more convenient wavelength from thestandpoint of sample transmission.

Examples of flooders especially useful with europium include salts, suchas potassium carbonate, sodium tungstate, and the like, and organicmaterials such as phenanthroline.

Except in cases where the concentration of species-linked diaminetriacid is high, such as 10⁻¹ to 10⁻⁴ M, in which case flooderconcentration will be similar, the amount of flooder used is very largerelative to the amount of species-linked diamine triacid. It isgenerally preferred to use at least 10³ times as much flooder as metalion or diamine triacid, on a molar basis with molar excesses of from 10³to 10¹⁰ being more preferred. These gross excesses are called forbecause the stability of the flooder-rare earth complexes is many ordersof magnitude lower than the stability of the complex between the diamineand the rare earth metal.

The flooded complex is formed simply by adding the flooder to thealready-formed rare earth chelate complex in solution. This is generallycarried out immediately before measuring the fluorescence of the floodedcomplex. Although not known with certainty, it appears that the floodedcomplex that forms is a 1:1:1 molar ternary combination ofspecies-linked diamine triacid:rare earth metal ion:flooder.

PREPARATIVE METHODS

The species-linked diamine triacids may be simply formed by contacting aspecies substance (which substance must contain at least one ##STR8##group) with a substantial molar excess of a diamine tetraacetic aciddianhydride of the formula shown in General Formula III in liquid phasein a polar aprotic liquid organic reaction medium. Useful reaction mediainclude acetone, dimethylformamide (DMF), dimethylsulfoxide (DMSO) andHMPA, and the like. Mixtures of these materials may be used as well.When the reactive group on the species molecule is an amine, especiallya primary amine, water may be used as solvent as may mixed solventscontaining water.

This contacting is carried out under conditions that will permit theactive group on the species molecule to react with one of the twoanhydride groups on the diamine. In the case where the active group onthe species molecule is an amine, the contacting will effect reaction atmoderate conditions, such as at temperatures of from about 5° C. toabout 100° C. with temperatures of from about 10° C. to about 75° C.being preferred. In the case where the active group on the species is athiol or a hydroxyl, somewhat more strenuous conditions are generallyemployed such as temperatures from 25° C. to about 150° C. In this case,temperatures of from 35° C. to about 125° C. are preferred. Thisdifference in reactivity may be utilized to effect selective reactionwith amine groups and exclusion of reaction with thiols or hydroxyls, ifsuch is of advantage. The reaction times employed, of course, areinversely dependent upon the temperature used. Generally, times of fromabout 0.5 hours to about two days are employed, with times of from onehour to about thirty-six hours being preferred. These times andtemperatures are provided as guides. In certain situations, such as withextremely heat sensitive or heat insensitive targets, it may be ofadvantage to go outside of the exemplary ranges shown here. As a generalrule, the use of lower temperatures within these ranges is to bepreferred as they lead to better selectivity of the reaction between theanhydride and the species molecule.

It is essential that an excess of the dianhydride be employed.Dianhydride:species molecule ratios of at least 4:1 should be employedwith ratios of from 5:1 to about 10:1 being preferred. The upper limiton this ratio is an arbitrary one based on a desire for economy. One mayuse amounts of anhydride greater than called for by this range andachieve good results. Such a mode of operation is considered to bewithin the scope of this invention but is also considered to be wastefulas the excess anhydride is destructively hydrolyzed in the workup whichfollows.

The dianhydrides employed herein are either art-known materials (seeU.S. Pat. No. 3,497,535) or may be prepared from the correspondingdiamine tetraacetic acids by the usual methods for preparing carboxylicdiacid anhydrides such as by heating the tetraacid to 150° C. orthereabouts (say 80° C. to 180° C.) in the presence of a molar excess ofacetic anhydride and a tertiary amine for a prolonged period such as forfrom 12 to 36 hours.

Following attachment of the species molecule to the dianhydride, theremaining anhydride group is hydrolyzed to the corresponding diaceticacid. This hydrolysis is easily carried out by admixing the couplingreaction mixture with an excess of water beyond the number of moles ofanhydride to be hydrolyzed and heating. Suitable temperatures are in therange of from about 30° C. to about 125° C. Suitable times are fromabout one minute to about two hours. Preferred temperatures and timesare in the ranges of 40° C. to 105° C. and two minutes to ninetyminutes. Alternatively, other conditions known in the art forhydrolyzing carboxylic acid anhydrides to acids (e.g., acetic anhydrideto acetic acid) may be employed.

The excess over stoichiometric of anhydride which is employed to formthe species-linked triacid is separated from the species-linkedmaterial. This removal of the excess anhydride can take place eitherbefore or after hydrolysis of the anhydride to acid as is found to bemost convenient. If the excess is not removed it will be available toreact with metal ions and optional flooders if present and likely giverise to complexes which will interfere with the precise determination ofthe amount of species-linked diamine triacid. This separation of linkeddiamine from nonlinked can be effected by any method which willdiscriminate between the species-linked and nonlinked molecules. The twospecies differ in size so that chromatography techniques such as gelpermeation chromatography, high pressure liquid chromatography, columnchromatography, for example, using a silica gel substrate, or thin layerchromatography can be used. Membrane techniques including dialysis andultrafiltration may also be used to effect this separation. In manycases, the linked material may differ from the nonlinked anhydride oracid by some physical property such as electrical charge or solubility.Such a property difference can also be used as a basis for separation,for example, by selective extraction or electrophoresis. Although wehave employed and generally prefer the chromatographic separations, theexact method employed should be based on the actual materials andseparation involved. The species-linked diamine triacid, freed of excessanhyride or acid is then formed into the metal complex.

The hydrolysis generates the desired species-linked diamine triacids ofFormula I. These may be converted to the metal coordination compounds ofFormula II by contacting the triacid with the desired metal ion. Thiscontacting is generally carried out in solution. The amount of metal ionemployed should be about one mole of ion per mole of triacid. Thecomplex between the metal ion and the triacid is a strong one havinglarge stability constants. This means that it is not required to employgross excesses of metal ion to drive the complex-forming reaction andexcesses of ion can in some cases interfere with the accuracy of laterfluorescence measurements.

The metal complex-forming step is generally carried out in an aqueousreaction medium. The water-containing hydrolysis reaction medium can bevery suitably employed, if desired. This means that the metal ions aregenerally added in the form of their water-soluble salts such ashalides, nitrates, acetates, or the like, as is appropriate.

The addition of metal ion is suitably carried out at ambient conditions.Complex formation is not favored by highly elevated temperatures andsignificantly depressed temperatures are not seen to offer anyadvantages. A temperature range of from 5° C. to about 40° C. isgenerally preferred for convenience.

In the case of the rare earth metal ion complexes for fluorometric assaytechniques it is often desired to add a "flooder" to the metal ioncomplex. This addition of flooder is usually carried out immediatelyprior to the measurement of the fluorescence. It is accomplished byadding the excess of flooder to the solution of the metal ion complex.This addition is carried out at moderate conditions as well, such as ata temperature of from 5° C. to 40° C.

SPECIES MOLECULES

Species molecules which may be covalently incorporated into the presentspecies-linked diamine triacid materials and complexes of this inventionare organic molecules selected from "target molecules" as thesematerials are described in above-noted U.S. Pat. Nos. 4,150,295 and4,058,732, which are incorporated by reference, molecules essentiallyidentical to "target molecules" and antibodies specific to "targetmolecule". These species molecules contain at least one of the groupsprimary amine, secondary amine, thiol, or hydroxyl. These active groupsmay be present intrinsically on the species molecule or they may bepresent on a "spacer" unit covalently linked to the species molecule.The spacer is of utility to minimize the possibility of interferencebetween the diamine triacid group and the species molecule itself. Suchinterference could be detrimental in certain situations, such as whenthe species molecule must exhibit immune reactive properties or takepart in some other substrate recognition step. The "spacer" may also beemployed just to provide the needed amine, thiol or hydroxyl activegroups. The term "species" includes materials with and without addedspacers.

The use of spacer or "bridge" molecules to attach active molecules tononactive groups, such as substrates has been developed in the fields ofenzyme immobilization, chromatography, and the like. See, for example,U.S. Pat. No. 3,278,392 of Patchornik, U.S. Pat. No. 3,873,514 of Chu etal, and Wilchek, FEBS LETT, 33 (1), 70-72. These techniques, while notsetting out the exact systems herein involved, do disclose a wide rangeof reagents and the advantages to be derived by the use of spacers.

Although this invention can be practiced with any species molecule whichinherently, or through the use of a spacer, contains the required amine,thiol or hydroxyl group, it finds especially advantageous applicationwith biologically active target molecules including therapeutic drugs,enzymes, hormones, peptides, macromolecules including proteins andlipids, haptens, antigens, and the like. Such molecules usually have atleast one of the required active groups and often present difficult oreven impossible analysis or detection problems because of the minuteamounts in which they may be present in biological systems.

Examples of the species molecules thus can range from simple organictarget molecules having the requisite functional groups, for example,lower alkanols--methanol, ethanol, butanol, hexanol, cyclohexanol, andthe like; lower aromatic hydroxy compounds--phenol, 2,4-dinitrophenoland the cresols; lower alkyl and aromatic amines--ethylamine,diethylamine, butylamine, and isopropylamine; lower thiol--propane thioland butane thiol; as well as targets up to the more complicated drug andbiological molecules including the hormones thyroxine, triiodothyronine,human growth hormone gonadotropins, gastrointestinal hormones, insulin,and the like; therapeutic drugs such as digoxin, morphine, procain amideand the like; proteins, antibodies and the like. In the case of largemolecules such as proteins and antibodies, there may be more than one(even a large number) of the requisite functional sites on each speciesmolecule so that each species molecule may have more than one diaminetriacid group covalently attached thereto. This list of possible speciesmolecules is to demonstrate the wide range of targets that can be linkedto the diamine triacids in accord with this invention. It is notintended to limit the scope of this invention.

ANALYSIS TECHNIQUES

As already described, the present invention enables the covalentattachment of complexes of metal ions to species molecules or toantibodies of species molecules. Detection of these metal ions bymethods known to the art can be used to determine the presence or amountof the species molecules. As also already mentioned, if the metal ionincorporated is a radionucleotide, radioassay techniques can be used todetermine the amount of species present. Alternatively, atomicabsorption techniques can be used to measure the amount of metal and theamount of target thus determined.

In one preferred embodiment the metal is a rare earth and the resultingchelate is activated to fluorescence by a flooder or other means. Thenumerous techniques which have evolved in the radioimmunoassay field canin general be applied to fluorescence immunoassay. These includeincubation techniques, competitive binding methods and separations ofbound and free labeled targets as discussed, for example, in the textsRadioimmunoassay And Related Techniques by J. I. Thoreu et al, C. V.Mosby Co., St. Louis, Mo., (1978) and Radioimmunoassay in ClinicalBiochemistry, Ed., C. A. Pasternak, Heyden, London, New York, Rheine,(1975).

The primary difference between radioimmunoassay and fluorescenceimmunoassay is in the final reading step. In the former case,radioactive disintegrations are counted, in the latter case,fluorescence is excited and measured.

Preferably, the time-gated fluorescence techniques disclosed in U.S.Pat. Nos. 4,150,295 and 4,058,732, both of Irwin Wieder, are employed asthey permit substantially enhanced sensitivity to be achieved.

In the case of fluorescent antibodies tagged with the rare earthchelates of the present invention, they can be used to assay for freetargets in the body fluids or in another preferred application they canbe used to detect the presence of (or to quantify) specific kinds ofbiological cells or bacteria provided that these cells or bacteria haveunique targets or groups of targets on their surface.

The invention will be further shown by the following Preparations andExamples. These are intended to exemplify the invention and are not tobe construed as limiting its scope which is instead defined by theappended claims.

PREPARATION OF DIAMINE TETRAACETIC ACID DIANHYDRIDES (a) EthylenediamineTetraacetic Acid Dianhydride

A mixture of ethylenediamine tetraacetic acid (EDTA) 364 g, 510 g ofacetic anhydride and 600 g of pyridine are heated to 65° C. and thereheld for 24 hours. The mixture is then cooled and filtered in a glovebox. The solid that is recovered is washed with diethyl ether and dried.It amounts to 96% of the theoretical weight of EDTA dianhydride.Elemental analysis confirms that this dianhydride is the productobtained.

(b) Propylenediamine Tetraacetic Acid Dianhydride

Following the procedure set forth in U.S. Pat. No. 3,660,388 of Dazzi,92 g of 1,3 propylenediamine tetraacetic acid, 152 g of acetic anhydrideand 113 g of pyridine are stirred for 24 hours at 65° C. The reactionproduct is evaporated to dryness under vacuum. The residue is recoveredrinsed three times with diethyl ether and dried in a vacuum oven at 100°C. The dried product is the desired dianhydride.

(c) Phenylene 1,2-diamine Tetraacetic Acid Dianhydride

Following the procedure of Dazzi, 34 g of 1,3-phenylenediaminetetraacetic acid, 72 g of acetic anhydride and 39 of pyridine arestirred for 24 hours at 65° C. The mixture is cooled and filtered. Thesolid that is separated is washed with benzene and dried. It is thedesired dianhydride.

EXAMPLE 1 A. Bonding Thyroxine to EDTA Dianhydride

A 100 ml flask is evacuated and flame dried followed by three cycles ofevacuation and filling with dry argon. The flask is cooled and chargedwith 433 mg of EDTA dianhydride, which is produced as in thePreparations, and 300 mg of L-thyroxine sodium salt pentahydrate. Thisthyroid gland hormone is of fundamental importance being vital fornormal growth and metabolism and may be obtained commercially. The moleratio of dianhydride:hormone is 5:1. Addition of 10 ml of DMF gives alight yellow solution. The flask is foil covered and warmed to 55° C.for 20 hours. A spot test is used to show that all of the hormone hasreacted with the dianhydride to give the product ##STR9##

B. Hydrolyzing the Residual Anhydride

Product (A) is not isolated. Instead, 4 ml of distilled water is addedto the flask and the heating is continued for an additional 1.5 hours.This causes the remaining anhydride group to hydrolyze yielding thethyroxine-linked ethylenediamine triacetic acid. The triacid and theEDTA that form are recovered as solids. The EDTA is recovered byfiltration, washed with DMF and dried. The triacid passes through thefilter and is recovered by evaporation of the filtrate.

C. Separating the EDTA from the Triacid

The crude product of Part (B) is eluted through a packed silica gelcolumn using as eluent ethanol:water (95:5) followed by 80:20 95%aqueous ethanol:30% aqueous ammonia. The triacid elutes with the aqueousethanol:aqueous ammonia and is collected as a pure compound by tlc. NMR,elemental analysis and IR scans verify that the recovered product is thedesired thyroxine-linked triacid (C) as the ammonium salt in 59% yield.

D. Forming a Complex of C with a Rare Earth Ion

A known amount of the thyroxine-linked diamine triacetic acid recoveredin part C is weighed out and dissolved in a few ml of 0.1 N NaOH. Withina few minutes, a stoichiometric quantity of a rare earth halide(specifically in this Example terbium chloride) is added and mixed toform a homogeneous solution containing the desired rare earth (terbium)ion/thyroxine-linked diamine triacetic acid 1:1 complex (D) and sodiumchloride which is not an interfering factor.

E. Forming a Ternary Complex of D with a Flooder

An aliquot of the terbium complex of Part D is placed in 10 ml of waterto give a concentration of about 10⁻⁸ molar. 5-sulfosalicylic acid in aconcentration of 10⁻² molar is added to form a terbium:thyroxine-linkeddiamine triacid:flooder complex. When excited at about 3300 Å, thiscomplex exhibits a strong fluorescence which can be detected at 5450 Å.The application of this fluorescence property to the assay of thyroxineis shown hereafter.

EXAMPLE 2

The preparation of Example 1 is repeated with two changes. In place ofterbium chloride in Step D, europium chloride is employed.Phenanthroline is used in place of 5-SSA as flooder. This results in theformation of the europium chelate and ternary complex corresponding tothe terbium complexes formed in Example 1. The europium complex isfluorescent when used with a phenanthroline flooder and permits thedetermination of the amount of thyroxine present in solutions in accordwith the method shown hereafter.

EXAMPLE 3 A. Bonding Cholesterol to EDTA Dianhydride Plus Hydrolysis

A 10 ml round bottomed flask is dried by flame heating and then filledwith argon. It is then charged with 200 mg (0.52 mmol) of recrystallizedcholesterol and 662 mg (2.59 mmol) of the dianhydride of Preparation a).The flask is left under vacuum for 45 minutes and then 3 ml of dried DMFis added and the mixture held at 90° C. for 36 hours. During this periodthe mixture remains essentially homogeneous and colorless. It is cooledto room temperature and 3 ml of deionized water is added with additionalcooling. A precipitate forms and the heterogeneous mixture is stirredfor 10 hours.

The mixture is diluted with 40 ml of diethyl ether-acetone (1:4) whichresults in additional precipitation. The mixture is filtered and thesolid washed with 10 ml of the ether-acetone mixture. The filtrates arecollected, evaporated and diluted with additional ether to cause moresolid to form. This is collected and added to the previously collectedsolid. After drying, 322 mg (48% of theory) of the desiredN-(ethanol-O-cholesterol)ethylene-diaminetriacetic acid. Analysis by NMRand TLC shows that the product is very pure. Elemental analysis isconsistent with the desired compound.

B. Forming Metal Ion Complex

The material of Part A (30 mg) is dissolved in 3 ml of 0.1 N NaOH. Arare earth metal ion (either terbium or dysprosium) is added in anapproximately stoichiometric amount (1.00 to 1.05 moles per mole of thecompound of Part A). This causes a rare earth metal ion complex of thematerial of Part A to form. This material, when promoted by the additionof an excess of a flooder exhibits fluorescence properties useful influoroassay methods. This addition of flooder is carried out as setforth in Example 1.

EXAMPLE 4 Coupling a Simple Alkanol

Isopropanol (1 mmol) is admixed with 10 ml of dried DMF in accord withthe method of Example 3. Five mmols of the dianhydride of Preparation(a) is added and the mixture is held at 90° C. for 24 hours. This causesthe alkanol's hydroxyl group to react with the anhydride and couple thealkanol in an ester configuration. This ester can be separated from theresidual anhydride either before or after hydrolysis of the anhydrideunits. Complexes can be formed by admixing the ester with the desiredmetal ion.

EXAMPLE 5 Coupling a Simple Amine

The preparation of Example 4 is repeated with two changes. First,ethylamine is substituted for isopropanol on an equimolar basis. Second,the reaction conditions are less stressful, a temperature of 35° C.being employed. The ethylamine molecules add to the dianhydride in anamide configuration.

EXAMPLES 6-8 Coupling of Other Simple Groups

The coupling of Example 4 is repeated substituting phenol, ethylthiol,and diethylamine for the isopropanol employed in Example 4. In the caseof diethylamine a lower temperature (35° C.) is employed as well. Theserepeats give rise to the coupled products based on each of the newstarting materials. They may be further processed to form metal ioncomplexes, if desired.

EXAMPLE 9 Coupling an Amine in an Aqueous Environment

The coupling of Example 5 is repeated with two additional changes. Inplace of the DMF solvent, water is used and in place of the 35° C. for24 hours, 30° C. and 36 hours is used. At these mild conditions the moreactive amine groups react and there is preferential attachment of theamines to the dianhydrides to form the desired triacids. This reactionis similar to the preferential reaction of an amine with aceticanhydride in an aqueous environment (see Caldwell, et al, J.A.C.S., 64,1695, (1942). This is a preferred method to couple triacids to proteinsand antibodies.

EXAMPLE 10 A. Bonding Thyronine to PDTA Dianhydride

A dried flask is charged with 6 mmol of PDTA dianhydride, which isproduced as in the Preparations, and 1 mmol of the essential amino acidthyronine, obtained from a commercial source. The mole ratio ofdianhydride:thyronine is 6:1. DMF in the amount of 50 ml is added andthe solution that forms is warmed to 35° C. and there maintained for 30hours. The mixture is spot tested on a TLC plate and found to have nounreacted thyronine and to contain the desired thyronine-linkedpropylenediamine triacetic acid anhydride.

B. Hydrolyzing, Separating and Complexing

Following the general methods shown in Example 1, the anhydride of PartA. is hydrolyzed in situ and separated from the excess PDTA that formsby column chromatography. This yields the desired thyronine-linkedtriacetic acid. The solution of the desired triacid is divided intothree equal parts. The first (estimated to contain about 0.2 mmoles ofthe triacid) is admixed with 1-2 mmoles of cobalt chloride as an aqueoussolution. This gives rise to a cobalt chelate of the thyronine-linkedtriacetic acid. The second portion is contacted with 0.2 mmoles ofterbium in the form of a solution of terbium bromide. This gives rise toa terbium chelate of the triacid. In accord with the method of Example1, the flooder, 4-aminosalicylic acid, 1×10² mmoles, is added to formthe 1:1:1 terbium/triacid/flooder complex. The third portion iscarefully contacted with 1-2 mmoles of In¹¹¹ as its chloride. This givesrise to the In¹¹¹ complex of the thyronine-linked triacid.

EXAMPLE 11

The preparation of Example 5 is repeated with one change. Instead ofEDTA dianhydride, cyclohexylene-1,2-diamine tetraacetic acid dianhydrideis employed. This causes the ethylamine-linked triacetic acid to beformed. This compound, ##STR10## is contacted with europium ions to giverise to the europium complex which in turn is converted to afluorescently active complex by addition of a 10⁸ molar excess of theflooder, potassium carbonate. This complex is detectable by fluorescencetechniques.

Application of Target-linked Diamine Triacid Complexes to Assay ofTarget Molecules

As a first step, the fact that fluorescence of a species-linked diaminetriacid-rare earth metal ion complex is related to concentration of thecomplex is demonstrated. A serial dilution of a solution of thethyroxine-linked diamine triacetic acid complex with terbium (preparedas in Part D. of Example 1) is carried out with concentrations of thethyroxine of from 10⁻⁶ molar to 2.5×10⁻¹¹ molar. Flooder is added (10⁻²molar 5-sulfosalicylic acid). At pH 11.5, fluorescence of each of thesamples is measured using a time-gated fluorimeter and measuringtechniques set forth in Immunofluorescence and Related StainingTechniques, Knapp, et al, eds., 67-80, Elsevier/North Holland BiomedicalPress, Amsterdam, 1978. The signals are plotted on log--log paper andseen to be linear over this range. The curve can be extrapolated toabout 10⁻¹² molar as a threshold detection limit. A threshold ofdetection is that concentration at which the fluorescent signal is justequal to the fluctuation in signals from a blank sample. The signalsfrom a blank sample include photomultiplier dark noise and residualbackground signals from sample container, solvents, buffers, and thelike. The fact that a linear plot is obtained shows that theconcentration of a tagged species can be determined by measuring itsfluorescence.

This finding is put to practical use, determining the concentration ofthyroxine in unknown concentration samples in plasma. First, a standardcurve is generated along the lines as in radioimmunoassay. A serialdilution of nonspecies-linked thyroxine is prepared in thyroxine-freeplasma. Thyroxine-free plasma is made according to art-known techniquesincluding denaturation of all thyroxine-bonding plasma proteins. Theconcentrations are 1, 2, 4, 8, and 16 μg/dl. pH is adjusted and a setamount of complex-linked thyroxine (D of Example 1) is added to eachdilution along with a set amount of antibody to thyroxine. This mixtureis incubated at conventional competitive binding conditions. Theantibody-bound and free thyroxine are then separated using any of theart-known separation steps of ammonium sulfate precipitation, doubleantibody precipitation, polyethylene glycol precipitation,dextran-charcoal precipitation, column separation, or the like. Thebound fraction is then suspended in buffer, flooder is added andfluorescence is measured using the time-gated process previously setforth. The observed fluorescence is plotted versus the concentration ofthe unlinked thyroxine in the standard samples. A curve is generatedconfirming that at larger concentrations of unlinked thyroxine thelinked material is less able to compete for antibody sites and theobserved fluorescence is lower. The curve that is generated is then usedto determine the concentration of thyroxine in unknown samples.

Serum samples containing unknown amounts of thyroxine are assayed fortotal thyroxine. First they are acidified to denature any bondingproteins and free the thyroxine from any attachment. Then, after pHadjustment, they are treated with a standard amount of thethyroxine-linked diamine triacid metal complex of Part D., Example 1,and a standard amount of thyroxine antibody as was done in thegeneration of the standard curve. The mixture is incubated as above.Bound and unbound materials are separated. Flooder is added and thefluorescence is measured. The concentration of thyroxine in the unknownis then read from the standard curve based on the observed fluorescence.

Application of Antibody-linked Diamine Triacid Complexes to Assays A.Assay for Antibody

In some cases the target molecule (molecule to be detected or measured)is an antibody. For example, it may be medically important to determinethe spectrum of the immunoglobulin (IgE) antibodies in a human patientsuffering from allergy. The patient may have IgE antibodies to variousallergens and in different quantities, and for proper treatment it iscrucial to know this distribution. To measure the amount of IgEantibodies in the blood or other body fluid the allergen in question iscovalently coupled to a solid phase substrate using known art techniquesand the substrate is exposed to the body fluid of the patient. After asuitable incubation period under suitable conditions the substrate isremoved and washed. A representative amount of IgE antibody specific tothe allergen on the solid substrate will now be present on thesubstrate, bound to the allergen. The substrate is now exposed toterbium chelated diamine triacid-linked antibody to human IgE; this antiIgE antibody is, in general, specific to all sub-classes of human IgEregardless of the allergen involved. After the second exposure, againunder suitable conditions the solid substrate is again washed andexposed to a flooder such as sodium tungstate. The fluorescence is thenmeasured preferably using a time-gated fluoromide as described above.Using known art procedures standard curves can be generated for eachallergen with known amounts of IgE and the unknown amount of IgE in ahuman sample can thus be determined by comparison with a standard curve.

B. Assay for Cells or Bacteria

Cells or bacteria can have various molecular markers on their surfaces.These markers can, in some cases, be identified, isolated or purifiedand can be used to produce antibodies specific to the marker. Suchantibodies, when linked to the diamine triacid metal complex of thisinvention can be used to identify certain cells or bacteria that havethe markers on their surface. The cells or bacteria are exposed to thediamine triacid metal linked-antibodies and if the marker is present, arelatively high concentration of linked antibody is bound to thesurface. These cells are then examined in a fluorescent microscope orfluorescent cell flow system and the fluorescent cells or bacteria arecounted for a given volume, thus revealing their presence in aquantitative or qualitative fashion.

These are but two examples of the many possible assays that can becarried out using the species-linked diamine triacetic acid complexes ofthe present invention. Other equivalent procedures may be employed, aswell.

What is claimed is:
 1. A metal chelate for use in the fluorometricquantification of a biologically active organic species comprising anessentially 1:1 molar chelate complex of a rare earth metal ion capableof forming a fluorescent complex with a species-linked diamine triaceticacid having the structural formula: ##STR11## wherein R is a two toeight atom long covalent bridge selected from carbon-oxygen etherbridges, carbon-nitrogen secondary and tertiary amide bridges andcarbon-carbon alkylene, cycloalkylene and arylene bridges, T is thebiologically active organic species to be fluorometrically quantified, Tbeing other than an aliphatic mono- or polyamine, or aliphatic mono- orpolyol, but comprising at least one originally present or added primaryamine, secondary amine, thiol or hydroxyl functional group, and L issaid at least one functional group in deprotonated form covalentlybonding T to the indicated carbonyl carbon of the diamine triaceticacid.
 2. The metal chelate of claim 1 wherein the rare earth metal ionis selected from among ions of terbium, dysprosium, europium, samariumand neodimium.
 3. The metal chelate of claim 1 wherein R is selectedfrom two to eight atom long carbon-oxygen ether bridges and two to fourcarbon atom long alkylene bridges.
 4. The metal chelate of claim 3wherein the rare earth metal ion is selected from among ions of terbium,dysprosium, europium, samarium and neodimium.
 5. The metal chelate ofclaim 1 wherein T is a therapeutic drug.
 6. The metal chelate of claim 1wherein T is an enzyme.
 7. The metal chelate of claim 1 wherein T is ahormone.
 8. The metal chelate of claim 1 wherein T is a macromolecularspecies.
 9. The metal chelate of claim 1 wherein T is an antibody. 10.The metal chelate of claim 1 wherein T is a lipid.
 11. The metal chelateof claim 1 wherein T is an antigen.
 12. The metal chelate of claim 1wherein T is a hapten.
 13. The metal chelate of claim 1 wherein R isselected from the group consisting of ethylene, n-propylene,isopropylene, n-butylene, 1- and 2-methylpropylene, 1-propylethylene,1-cyclohexylethylene, 1-phenylethylene, alkyl substituted1-phenylethylenes and propylenes, 1-benzylethylene, 2-amidopropylene,cyclohexyl-1,2-ene, phenyl-1,3-ene, --CH₂ --CH₂ --O--CH₂ --CH₂ --, and--CH₂ --CH₂ --O--CH₂ --CH₂ --O--CH₂ --CH₂ --.
 14. The metal chelate ofclaim 3 wherein the metal ion is selected from among the ions ofeuropium and terbium.
 15. The metal chelate of claim 14 wherein R is atwo carbon atom long alkylene bridge.
 16. The metal chelate of claim 15wherein the metal ion is terbium ion.
 17. An activated fluorescent rareearth metal ion chelate combination for use in the fluorometricquantification of a biologically active organic species comprising anessentially 1:1 molar chelate complex of a rare earth metal ion capableof forming a fluorescent complex with a species-linked diamine triaceticacid having the structural formula: ##STR12## wherein R is a two toeight atom long covalent bridge selected from carbon-oxygen etherbridges, carbon-nitrogen secondary and tertiary amide bridges andcarbon-carbon alkylene, cycloalkylene and arylene bridges, T is thebiologically active organic species to be fluorometrically quantified, Tbeing other than an aliphatic mono- or polyamine, or aliphatic mono- orpolyol, but comprising at least one originally present or added primaryamine, secondary amine, thiol or hydroxyl functional group, and L issaid at least one functional group in deprotonated form covalentlybonding T to the indicated carbonyl carbon of the diamine triacetic acidin combination with a fluorescent excitation efficiency promoter. 18.The activated fluorescent chelate combination of claim 17 wherein therare earth metal ion is selected from among ions of terbium, dysprosium,europium, samarium and neodimium.
 19. The activated fluorescent chelatecombination of claim 17 wherein R is selected from two to eight atomlong carbon-oxygen ether bridges and two to four carbon long alkylenebridges.
 20. The activated fluorescent chelate complex of claim 19wherein the rare earth metal ion is selected from among ions of terbium,dysprosium, europium, samarium and neodimium.
 21. The activatedfluorescent chelate complex of claim 17 wherein T is a therapeutic drug.22. The activated fluorescent chelate complex of claim 17 wherein T isan enzyme.
 23. The activated fluorescent chelate complex of claim 17wherein T is a macromolecular species.
 24. The activated fluorescentchelate complex of claim 17 wherein T is an antibody.
 25. The activatedfluorescent chelate complex of claim 17 wherein T is an antigen.
 26. Theactivated fluorescent chelate complex of claim 17 wherein T is a lipid.27. The activated fluorescent chelate complex of claim 17 wherein T is ahapten.
 28. The activated fluorescent chelate complex of claim 17wherein the fluorescent excitation efficiency promoter is a salicylate.29. The activated fluorescent chelate complex of claim 27 wherein themetal ion is terbium ion, R is a two carbon atom long alkylene bridgeand the fluorescent excitation efficiency promoter is 5-sulfosalicylicacid.
 30. The process for preparing a fluorescent species-linked diaminetriacetic acid which comprises the steps ofa. contacting in liquid phasea biologically active organic species molecule having a functional groupselected from ##STR13## but being other than an aliphatic mono- orpolyamine, or an aliphatic mono- or polyol with a molar excess of adiaminedianhydride of the formula ##STR14## wherein R is a two to eightatom long covalent bridge selected from carbon-oxygen ether bridges,carbon-nitrogen secondary and tertiary amide bridges and carbon-carbonalkylene, cycloalkylene and arylene bridges, per mole of speciesmolecule at a temperature of from about 5° C. to about 100° C. for fromabout 0.5 hours to about two days, b. hydrolyzing the remaininganhydride groups, c. recovering the species-linked diamine triaceticacid which is formed in step b, d. contacting the recoveredspecies-linked diamine triacetic acid with a solution of a rare earthmetal ion capable of forming a fluorescent complex with said triaceticacid and e. contacting said fluorescent complex with a substantial molarexcess of a fluorescent excitation efficiency promoter.